Otitis media with effusion (OME) is a significant health problem of children. Haemophilus influenzae is one of the major causes of this disease. Features of H. influenzae OME include frequent recurrences and a failure of eradication with antibiotic administration. One hypothesis explaining these features of H. influenzae OME is that the organism is growing as a biofilm in the middle ear. Bacterial biofilms are characteristically insensitive to antibiotic treatment, as well as incapable of elimination by the host inflammatory response. Evidence of a H. influenzae biofilm in children with OME consists of the presence of short-lived, Haemophilus-specific mRNA in the middle ear fluid of these children. Gram-negative bacterial biofilm formation is dependent upon the synthesis of quorum-sensing transcriptional activators, called autoinducers. We have found that H. influenzae; including those isolated from the middle ear possess a gene (HI0491) capable of synthesizing an autoinducer (AI-2). Insertional inactivation of HI0491 in the H. influenzae laboratory strain Rd KW20 results in a mutant, which lacks the ability to form mature biofilm structures, and has decreased susceptibility to antibiotics, a decreased conjugation frequency and decreased survival in an animal model of OME. In this application, we are seeking to characterize biofilm formation by several prototypic "otitic" H. influenzae in vitro, assess experimental OME caused by these strains in the weanling rat and chinchillas, determine the role of the autoinducer in this disease and define the role of AI-2 in biofilm development in vitro and in vivo. Understanding the role of AI-2 and biofilm formation in OME will permit strategies to prevent or treat this disease to be devised.